1. Field of the Disclosure
The present invention relates generally to certain Helicobacter pylori proteins, to the genes which express these proteins, and to the use of these proteins for diagnostic and vaccine applications.
2. Brief Description of Related Art
Helicobacter pylori is a curved, microaerophilic, gram negative bacterium that has been isolated for the first time in 1982 from stomach biopsies of patients with chronic gastritis, Warren et al., Lancet i:1273–75 (1983). Originally named Campylobacter pylori, it has been recognized to be part of a separate genus named Helicobacter, Goodwin et al., Int. J. Syst. Bacteriol. 39:397–405 (1989). The bacterium colonizes the human gastric mucosa, and infection can persist for decades. During the last few years, the presence of the bacterium has been associated with chronic gastritis type B, a condition that may remain asymptomatic in most infected persons but increases considerably the risk of peptic ulcer and gastric adenocarcinoma. The most recent studies strongly suggest that H. pylori infection may be either a cause or a cofactor of type B gastritis, peptic ulcers, and gastric tumors, see e.g., Blaser, Gastroenterology 93:371–83 (1987); Dooley et al., New Engl. J. Med. 321:1562–66 (1989); Parsonnet et al., New Engl. J. Med. 325:1127–31 (1991). H. pylori is believed to be transmitted by the oral route, Thomas et al., Lancet i:340, 1194 (1992), and the risk of infection increases with age, Graham et al., Gastroenterology 100:1495–1501 (1991), and is facilitated by crowding, Drumm et al., New Engl. J. Med. 4322:359–63 (1990); Blaser, Clin. Infect. Dis. 15:386–93 (1992). In developed countries, the presence of antibodies against H. pylori antigens increases from less than 20% to over 50% in people 30 and 60 years old respectively, Jones et al., Med. Microbio. 22:57–62 (1986); Morris et al., N. Z. Med. J. 99:657–59 (1986), while in developing countries over 80% of the population are already infected by the age of 20, Graham et al., Digestive Diseases and Sciences 36:1084–88 (1991).
The nature and the role of the virulence factors of H. pylori are still poorly understood. The factors that have been identified so far include the flagella that are probably necessary to move across the mucus layer, see e.g., Leying et al., Mol. Microbiol. 6:2863–74 (1992); the urease that is necessary to neutralize the acidic environment of the stomach and to allow initial colonization, see e.g., Cussac et al., J. Bacteriol. 174:2466–73 (1992); Perez-Perez et al., J. Infect. Immun. 60:3658–3663 (1992); Austin et al., J. Bacteriol. 174:7470–73 (1992); PCT Publ. No. WO 90/04030; and a high molecular weight cytotoxic protein formed by monomers allegedly having a molecular weight of 87 kDa that causes formation of vacuoles in eukaryotic epithelial cells and is produced by H. pylori strains associated with disease, see e.g., Cover et al., J. Bio. Chem. 267:10570–75 (1992) (referencing a “vacuolating toxin” with a specified 23 amino acid N-terminal sequence); Cover et al., J. Clin. Invest. 90:913–18 (1992); Leunk, Rev. Infect. Dis. 13:5686–89 (1991). Additionally, the following is also known.
H. pylori culture supernatants have been shown by different authors to contain an antigen with a molecular weight of 120, 128, or 130 kDa, Apel et al., Aentralblat fur Bakteriol. Microb. und Hygiene 268:271–76 (1988); Crabtree et al., J. Clin. Pathol 45:733–34 (1992); Cover et al., Infect. Immun. 58:603–10 (1990); Figura et al., H. pylori, gastritis and peptic ulcer (eds. Malfrtheiner et al.), Springer Verlag, Berlin (1990). Whether the difference in size of the antigen described was due to interlaboratory differences in estimating the molecular weight of the same protein, to the size variability of the same antigen, or to actual different proteins was not clear. No nucleotide or amino acid sequence information was given about the protein. This protein is very immunogenic in infected humans because specific antibodies are detected in sera of virtually all patients infected with H. pylori, Gerstenecker et al., Eur. J. Clin. Microbiol. 11:595–601 (1992).
H. pylori heat shock proteins (hsp) have been described, Evans et al., Infect. Immun. 60:2125–27 (1992) (44 amino acid N-terminal sequence and a molecular weight of about 62 kDa); Dunn et al., Infect. Immun. 60:1946–51 (1992) (33 amino acids found in the N-terminal sequence and a molecular weight of about 54 kDa); Austin et al., J. Bacteriol. 174:7470–73 (1992) (37 amino acids found in the N-terminal sequence and a molecular weight of about 60 kDa). Austin et al. suggest that these are, in fact, the same protein with identical amino acid sequences at their N-terminus.
For examples of diagnostic tests based on H. pylori lysates or semipurified antigens, see Evans et al., Gastroenterology 96:1004–08 (1989); U.S. Pat. No. 4,882,271; PCT Publ. No. WO 89/08843 (all relating to compositions and assays containing the same having high molecular weight antigens (300–700 kDa) from the outer membrane surface with urease activity); EPO Publ. No. 329 570 (relating to antigenic compositions for detecting H. pylori antibodies having fragments of at least one fragment from the group 63, 57, 45, and 31 kDa).
The percentage of people infected by H. pylori, either in a symptomatic or an asymptomatic form, is very high in both developing and developed countries, and the cost of hospitalization and therapy makes desirable the development of both H. pylori vaccines and further diagnostic tests for this disease.